A comparative study on the preparation of immunoglobulin-galactosidase conjugates.
For the application of beta-D-galactosidase-immunoglobulin conjugates in the enzyme-linked immunosorbent assay (ELISA), four techniques for the preparation of such conjugates were compared. Sheep immunoglobulin (Ig) (against soluble egg antigens of the trematode Schistosoma mansoni) was coupled to beta-D-galactosidase by means of 1) glutaraldehyde treatment, 2) the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and 3,4) two different procedures using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS). The prepared conjugates were then fractionated by gel filtration on Sepharose 6B and the resultant molecular weight fractions were tested in an ELISA for the detection of S. mansoni antigen. Optimal results were obtained with a conjugate that was synthesized according to one of the two techniques using MBS. With this conjugate, 10(-9) g antigen/ml could still be detected in an ELISA with a chromogenic substrate, which was at least ten times as sensitive as with the other conjugates. Application of a fluorogenic substrate resulted in a lower detection level of 10(-10) g antigen/ml.
D000818:Animals; D000941:Antigens / Q000032:analysis; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005696:Galactosidases; D005976:Glutaral; D007136:Immunoglobulins / Q000302:isolation & purification*; D007202:Indicators and Reagents; D011725:Pyridines; D012550:Schistosoma mansoni / Q000276:immunology; D012756:Sheep; D013388:Succinimides; D001616:beta-Galactosidase
D000941:Antigens; D007136:Immunoglobulins; D007202:Indicators and Reagents; D011725:Pyridines; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D005696:Galactosidases; D001616:beta-Galactosidase; D005976:Glutaral